Ccr Gene Complex Research Articles

Characterization of staphylococcal cassette chromosome mec in Staphylococcus haemolyticus ST3 and ST42 isolates from bovine mastitis milk: identification of novel variant in class C1 mec complex.

Methicillin-resistant Staphylococcus haemolyticus (MRSH) is an important pathogenic agent of bovine mastitis. Among the prominent clone lineages in dairy cows are MRSH sequence types ST3 and ST42. Little information is available on the complete characterization of SCCmec elements in MRSH. In this study, two clinical isolates of MRSH ST3 and ST42 from bovine mastitis milk were selected, and their nontypable SCCmec structures were compared. Two MRSH strains, MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1, were identified from bovine mastitis milk in Thailand in 2022. Minimum inhibitory concentration was used to screen for antimicrobial resistance susceptibility. Oxford Nanopore Technologies and Illumina sequencing were performed in combination to complete the genome. Their gene organization and structure of SCCmec types were analysed and compared with the whole sequences of other strains in the same sequence types. Both MRSH-ST3 strain M62.3 and MRSH-ST42 strain M81.1 possessed the class C1 mec complex but lacked the ccr gene complex. Notably, MRSH-ST42 strain M81.1 contained a novel variant of C1 mec complex, which consisted of IS431-mecA-ISSha1-paaZ-upgQ-IS431, with IS431 organized in the same orientation. Apart from class C1 mec and the heavy metal-resistant cluster, the gene composition and order of the SCCmec element varied. In ST3, variations in the SCCmec type, gene content and organization were observed. The distinct evolution of the MRSH lineage was indicated by the various SCCmec elements. The insertion of ISSha1 resulted in a unique variant of class C1 mec complex that demonstrated the important role of the insertion sequence in SCCmec diversification.

Unearthing New ccr Genes and Staphylococcal Cassette Chromosome Elements in Staphylococci Through Genome Mining.

Staphylococcal cassette chromosome mec (SCCmec) typing is crucial for investigating methicillin-resistant Staphylococcus aureus (MRSA), relying primarily on the combination of ccr and mec gene complexes. To date, 19 ccr genes and 10 ccr gene complexes have been identified, forming 15 SCCmec types. With the vast release of bacterial genome sequences, mining the database for novel ccr gene complexes and SCC/SCCmec elements could enhance MRSA epidemiological studies. In this study, we identified 12 novel ccr genes (6 ccrA, 3 ccrB, and 3 ccrC) through mining of the National Center for Biotechnology Information (NCBI) database, forming 12 novel ccr gene complexes and 10 novel SCC elements. Overexpression of 5 groups of novel Ccr recombinases (CcrA9B3, CcrA10B1, CcrC3, CcrC4, and CcrC5) in a mutant MRSA strain lacking the ccr gene and extrachromosomal circular intermediate (ciSCC) production significantly promoted ciSCC production, demonstrating their biological activity. This discovery provides an opportunity to advance MRSA epidemiological research and develop database-based bacterial typing methods.

Whole-Genome Investigation of Zoonotic Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolated from Pigs and Humans in Thailand.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been widespread globally in pigs and humans for decades. Nasal colonization of LA-MRSA is regarded as an occupational hazard to people who are regularly involved in livestock production. Our previous study suggested pig-to-human transmission caused by LA-MRSA clonal complex (CC) 398, using traditional molecular typing methods. Instead, this study aimed to investigate the zoonotic transmission of LA-MRSA CC398 using whole genome sequencing (WGS) technologies. A total of 63 LA-MRSA isolates were identified and characterized in Thailand. Further, the 16 representatives of LA-MRSA CC9 and CC398, including porcine and worker isolates, were subjected to WGS on the Illumina Miseq platform. Core-genome single nucleotide polymorphism (SNP)-based analyses verify the zoonotic transmission caused by LA-MRSA CC398 in two farms. WGS-based characterization suggests the emergence of a novel staphylococcal cassette chromosome (SCC) mec type, consisting of multiple cassette chromosome recombinase (ccr) gene complexes via genetic recombination. Additionally, the WGS analyses revealed putative multi-resistant plasmids and several cross-resistance genes, conferring resistance against drugs of last resort used in humans such as quinupristin/dalfopristin and linezolid. Significantly, LA-MRSA isolates, in this study, harbored multiple virulence genes that may become a serious threat to an immunosuppressive population, particularly for persons who are in close contact with LA-MRSA carriers.

Staphylococcus brunensis sp. nov. isolated from human clinical specimens with a staphylococcal cassette chromosome-related genomic island outside of the rlmH gene bearing the ccrDE recombinase gene complex.

Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.

Molecular characterization of typing and subtyping of Staphylococcal cassette chromosome SCCmec types I to V in methicillin-resistant Staphylococcus aureus from clinical isolates from COVID-19 patients.

Methicillin resistance is acquired by the bacterium due to mecA gene which codes for penicillin-binding protein (PBP2a) having low affinity for β-lactam antibiotics. mecA gene is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). SCCmec genomic island comprises two site-specific recombinase genes namely ccrA and ccrB [cassette chromosome recombinase] accountable for mobility. Currently, SCCmec elements are classified into types I, II, III, IV and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to variances in their J region DNA. SSCmec type IV has been found in community-acquired isolates with various genetic backgrounds. The present study was undertaken to categorize the types of SCCmec types and subtypes I, II, III, IVa, b, c, d, and V and PVL genes among clinical MRSA isolates from COVID-19 confirmed cases. Based on the Microbiological and Molecular (mecA gene PCR amplification) confirmation of MRSA isolated from 500 MRSA SCCmec clinical samples, 144 cultures were selected for multiplex analysis. The multiplex PCR method developed by Zhang et al. was adapted with some experimental alterations to determine the specific type of these isolates. Of the total 500 MRSA, 144 MRSA (60 were CA-MRSA and 84 were HA-MRSA) were selected for characterization of novel multiplex PCR assay for SSCmec Types I to V in MRSA. Molecular characterization of multiplex PCR analysis revealed results compare to the phenotypic results. Of the 60 CA-MRSA; in 56 MRSA strains type IVa was found and significantly defined as CA-MRSA while 4 strains showed mixed gens subtypes. Type II, III, IA, and V were present in overall 84 HA-MRSA. Molecular subtyping was significantly correlated to define molecularly as CA-MRSA and HA-MRSA however 15 (10%) strains showed mixed genes which indicates the alarming finding of changing epidemiology of CA-MRSA and HA-MRSA as well. We have all witnessed of COVID-19 pandemic, and its mortality was mostly associated with co-morbid conditions and secondary infections of MDR pathogens. Rapid detections of causative agents of these superbugs with their changing epidemiology by investing in typing and subtyping clones are obligatory. We have described an assay designed for targeting SSCmec types and subtypes I, II, III, IVa,V according to the current updated SCCmec typing system. Changing patterns of molecular epidemiology has been observed by this newly described assay.

Novel SCCmec variants in clonal complex 398 and lineage-specific pseudo-SCCmec identified in ST88 MRSA from invasive bloodstream infections in China.

Methicillin resistance in Staphylococcus aureus is primarily due to the mecA gene found in highly diverse staphylococcal cassette chromosome mec (SCCmec) elements, with an increasing number of variants being continually discovered. To characterize two novel SCCmec variants identified in clonal complex (CC) 398 strains and lineage-specific pseudo-SCCmec elements in the ST88 clone. WGS and comparative genomic analysis were used to elucidate the SCCmec element diversity of representative isolates. The non-typeable 47 kb SCCmec found in the CC398 strain SKLX55795 represents a novel subtype of XIV, showing significant differences in structural organization and genetic content within the joining regions compared with the XIV element from the prototype strain SC792. This unique subtype comprised remnants from various mobile genetic elements that encode antimicrobial resistance genes, ultimately forming a large MDR region. Genome analysis of CC398 strain SKLX61416 revealed the presence of a novel 50 kb composite SCCmec with two distinct domains, carrying the ccr gene complexes 5/8 and containing genes for the detoxification of arsenic and sulphide. Further sequence analysis disclosed that 44.23% (23/52) of ST88 strains in our collection carried a lineage-specific pseudo-SCCmec, termed ΨSCCmecST88. This ΨSCCmecST88 harboured the mec gene complex C2, along with a series of genes associated with heavy metal resistance, but lacked an approximately 28 kb region encompassing the ccr gene complex. Our findings provide evidence for the ongoing evolution of SCCmec elements within the CC398 and ST88 clones, underscoring the need for further surveillance to understand the biological significance of these elements.

Novel Organization of the Staphylococcal Cassette Chromosome mec Composite Island in Clinical Staphylococcus haemolyticus and Staphylococcus hominis Subspecies hominis Isolates from Dogs.

ABSTRACTStaphylococcus haemolyticus and Staphylococcus hominis subsp. hominis are common coagulase-negative staphylococcus opportunistic pathogens. In Thailand, the clinical strains S. haemolyticus 1864 and 48 and S. hominis subsp. hominis 384 and 371 have been recovered from sick dogs. These strains were methicillin resistant with the nontypeable staphylococcal cassette chromosome mec (NT-SCCmec). The SCCmec element distribution in the clinical isolates from dogs was analyzed using whole-genome sequencing, which revealed the presence of different SCCmec composite islands (CIs) and gene structure. The SCCmec-CIs of ψSCCmec1864 (13 kb) and ψSCC1864 (11 kb) with a class C1 mec complex but no ccr gene were discovered in S. haemolyticus 1864. The CIs of ψSCCmec48 with a C1 mec complex (28 kb), SCC48 with ccrA4B4 (23 kb), and ψSCC48 (2.6 kb) were discovered in S. haemolyticus 48. In SCC48, insertion sequence IS256 contained an aminoglycoside-resistant gene [aph(2″)-Ia]. Two copies of IS431 containing the tetracycline-resistant gene tet(K) were found downstream of ψSCC48. In S. hominis subsp. hominis, the SCCmec-CI in strain 384 had two separate sections: ψSCCmec384 (20 kb) and SCCars (23 kb). ψSCCmec384 lacked the ccr gene complex but carried the class A mec complex. Trimethoprim-resistant dihydrofolate reductase (dfrC) was discovered on ψSCCmec384 between two copies of IS257. In strain 371, SCCmec VIII (4A) (37 kb) lacking a direct repeat at the chromosomal end was identified. This study found SCCmec elements in clinical isolates from dogs that were structurally complex and varied in their genetic content, with novel organization.IMPORTANCE In Thailand, the staphylococcal cassette chromosome mec (SCCmec) element, which causes methicillin resistance through acquisition of the mec gene, has been studied in clinical coagulase-negative Staphylococcus isolates from various companion animals, and Staphylococcus haemolyticus and Staphylococcus hominis subsp. hominis were found to have the most nontypeable (NT)-SCCmec elements. These species are more prone to causing illness and more resistant to a variety of antimicrobials than other coagulase-negative staphylococci. However, full characterization of NT-SCCmec in clinical S. haemolyticus and S. hominis subsp. hominis isolates from such animals has been limited. Our findings support the use of full nucleotide sequencing rather than PCR designed for Staphylococcus aureus in further research of novel SCCmec elements. Moreover, several antimicrobial resistance and heavy metal resistance genes were identified on the SCCmec elements; these are important as they could limit the therapeutic options available in veterinary medicine.

Investigating the relation between resistance pattern and type of Staphylococcal cassette chromosome mec (SCCmec) in methicillin-resistant Staphylococcus aureus.

Background and Objectives:MRSA became a widely recognized cause of mortality worldwide with necessity of its epidemiological pattern study. Typing of MRSA isolates was performed molecularly based on SCCmec type and relation to resistance pattern was investigated.Materials and Methods:Out of 200 clinical specimens, S. aureus was detected phenotypically and confirmed as MRSA by PCR in 124 isolates obtained from associated laboratories of different hospitals of Zagazig, during 2018–2019. Antimicrobial resistance pattern was detected and MRSA SCCmec was typed by two methods.Results:S. aureus rate was high in wounds, sputum, blood, and urine isolates. Antimicrobial resistance rates against cefotaxime, tetracycline, gentamicin, ciprofloxacin, erythromycin, azithromycin, clindamycin, chloramphenicol, sulfamethoxazole-trimethoprim, linezolid and vancomycin were 82.3%, 65.3%, 56.4%, 45.1%, 37.1%, 32.3%, 32.3%, 25%, 7.3%, 2.4% and 0%, respectively. Multiplex-PCR(M-PCR) was able to detect SCCmec element among 57% of isolates classified as SCCmec II (n=40), III (n=21), IVa (n=3), IVd (n=2), V(n=1), and four isolates contain both SCCmec II and SCCmec IV. Traditional typing by PCR for mec and ccr gene complexes was almost concordant with M-PCR. Furthermore, it was able to identify SCCmec types VI, IX, and XIV among 1, 3 and 18 isolates, respectively. No Statistical correlation was established between type of cassette and rate of antimicrobial resistance. Phylogenetic analysis reveals that all ccr types were related to each other and no significant variation in the same ccr type of different SCCmec cassettes.Conclusion:Most MRSA isolates were MDR reflecting antimicrobials misuse. Detection of various SCCmec types among MRSA isolates indictae the complexity of MRSA epidemiology and increase chance for gene sharing creating new types. The presented investigation was important in understanding MRSA epidemiology and diversity in Egypt providing advice for improving therapeutic protocols.

Novel SCCmec type XV (7A) and two pseudo-SCCmec variants in foodborne MRSA in China.

Staphylococcal cassette chromosome mec (SCCmec) elements are highly diverse and have been classified into 14 types. Novel SCCmec variants have been frequently detected from humans and animals but rarely from food. To characterize a novel SCCmec type and two SCCmec variants identified from food-associated MRSA in China. Three MRSA (NV_1, NT_611 and NT_8) collected from retail foods in China were subjected to WGS and the SCCmec elements were determined. The novel SCCmecXV identified in NV_1 carried the mec gene complex class A (mecI-mecR1-mecA-IS431) and the ccr gene complex 7 (ccrA1B6), and a Tn558-mediated phenicol exporter gene fexA was detected in this SCCmecXV cassette. The pseudo-SCCmec elements ΨSCCmecNT_611 and ΨSCCmecNT_8 showed a truncated SCCmec pattern, carrying the class C2 mec gene complex but missing the ccr genes. The ΨSCCmecNT_611 element shared more similarities with those of Staphylococcus haemolyticus (AB478934.1) and carried a heavy metal resistance gene cluster cadD-cadX-arsC-arsB-arsR-copA. The ΨSCCmecNT_8 MRSA exhibited a highly resistant phenotype, showing the absence of a 19.3 kb segment compared with the reference SCCmecXII element (CP019945.1). Notably, a 46 kb region containing multiple transposons encoding antimicrobial or metal resistance genes flanked by IS431 or IS256 was identified ∼30 kb downstream from the mec gene complex in ΨSCCmecNT_8, which might explain such high resistance in MRSA NT_8. Our finding of novel and pseudo-SCCmec elements reflected the ongoing intra/interspecies genetic rearrangements in staphylococci. Further study will be needed to investigate the biological significance and prevalence of those SCCmec variants along the food chain.

Staphylococcal cassette chromosome mec elements from Staphylococcus intermedius group (SIG) isolates from dogs in a center for veterinary diagnostics in Brazil

ABSTRACT: Methicillin resistance in the Staphylococcus intermedius group (SIG) has emerged in small animal practice. Methicillin-resistant SIG (MRSIG) members have been implicated as causes of infections in both companion animals and humans. Staphylococcal cassette chromosome mec (SCCmec) elements carry the mecA/C genes, which encode for the transpeptidase PBP2a (PBP2’) responsible for β-lactam antibiotic resistance in staphylococci. This study examined the SCCmec types of MRSIG isolates from different clinical specimens of dogs that exhibited methicillin MIC ≥ 0.5 μg/mL by an automated identification and susceptibility system in a Center for Veterinary Diagnostics in São Paulo, Brazil. Susceptibility to methicillin was determined by broth microdilution testing, and Oxoid® M.I.C.Evaluator® strips. PBP2a production was detected using a latex agglutination assay. SCCmec typing was performed according to the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) guidelines. SCCmec type II (2A), SCCmec type III (3A), composite SCC structures consisting of a class A mec gene complex in addition to multiple ccr gene complexes, and non-typable SCCmec elements were reported in these MRSIG isolates. SCCmec type variants differing from those so far acknowledged by IWG-SCC were found, indicating new rearrangements in the genetic context of mecA in these canine MRSIG isolates.

Complete genome of a methicillin-resistant Staphylococcus vitulinus from Danish ground beef meat carrying a mecA2 resistance gene and a novel ccr allotype

ObjectivesTo report the complete genome sequence of a methicillin-resistant Staphylococcus vitulinus from ground beef to allow comparison with other available S. vitulinus genomes and to investigate its SCCmec element. MethodsMeat samples from grocery stores in Denmark were examined for the presence of staphylococcal species by plating on selective plates. One colony isolated from beef was identified as S. vitulinus by MALDI-TOF and genome sequenced using a combination of Illumina and Oxford Nanopore technologies. Phylogenetic and in silico resistome analyses were performed for all available S. vitulinus genomes. ResultsThe closed genome of S. vitulinus Tienloo1 isolate had a chromosome size of 2,628,028 bp and contained a single novel 2,380 bp plasmid based on a hybrid assembly. It carried mecA as the only resistance marker. The isolate was found not to carry any immune evasion cluster genes, which have been putatively associated to human origin. Comparison with all publicly available S. vitulinus draft genomes showed a diverse population and revealed that only the Danish beef isolate contained a mec gene in addition to a ccr gene complex. Additionally, the single ccrC gene within the isolate was novel and distant from the mecA2 gene. ConclusionThis isolate, Tienloo1, from a ground beef meat sample represents the first complete genome of S. vitulinus found to carry a mecA2 gene and a novel ccr allotype in its SCCmec element that is distinct from all publicly available draft S. vitulinus genomes.

Unconventional SCCmec types and low prevalence of the Panton-Valentine Leukocidin exotoxin in South African blood culture Staphylococcus aureus surveillance isolates, 2013-2016.

Staphylococcus aureus is a healthcare-associated pathogen that can harbour multiple antimicrobial resistance determinants and express multiple virulence factors e.g. Panton-Valentine Leukocidin (PVL). Unknown staphylococcal cassette chromosome mec (SCCmec) typing patterns were previously observed among 11% (n = 52) of methicillin-resistant S. aureus (MRSA) isolates; we further investigated these as well as the proportion of PVL, encoded by lukS/F-PV, in 761 S. aureus isolates from patients with a diagnosis of pneumonia/lower respiratory tract, skin/soft tissue, bone and joint infection. S. aureus isolates from blood culture were identified and antimicrobial susceptibility testing was performed using automated systems. Conventional PCR assays were used to identify the ccr and mec gene complexes in mecA-positive isolates with an unknown SCCmec type and screen for lukS/F-PV. Epidemiological data was used to classify isolates as healthcare- or community-associated infections. Antimicrobial susceptibility profiles according to SCCmec type and PVL were reported. Of the unknown SCCmec types, isolates were interpreted as type I-like (86%, 38/44), type II-like (9%, 4/44) and type III-like (5%, 2/44). Eight isolates did not produce definitive results. Of all MRSA isolates, majority were multidrug-resistant as indicated by their non-susceptibility to most antimicrobial agents; 92% were healthcare-associated. PVL was seen in 14% of the isolates (MRSA: 25%, MSSA: 75%); 56% were classified as healthcare-associated infection. The SCCmec typing method did not definitively classify all unknown isolates into clearly defined types. It showed that majority of these isolates were not the conventional types; untypeable elements appeared to be composite SCCmec elements, consisting of multiple ccr gene complexes. Majority of the MRSA isolates were non-susceptible to most antibiotics indicating that multiple resistance genes are present in our population. Furthermore, the proportion of PVL was low and more prevalent in MSSA.

Effects of gamma-irradiation on antibiotic resistance and diagnostic molecular markers of methicillin-resistant Staphylococcus aureus in Egyptian cancer patients

:Purpose: This in-vitro study aimed to assess in 120 [40 community-acquired (CA-MRSA) & 80 hospital-acquired (HA-MRSA)] isolates from cancer patients whether the transmissible staphylococcal cassette chromosome mec (SCCmec) typing, and the Panton–Valentine leukocidin (PVL) virulence genes detection could be employed as tools for molecular diagnostic purposes to distinguish both methicillin-resistant Staphylococcus aureus (MRSA) categories in radiotherapy treated cancer patients.Materials and methods: SCCmec typing was determined by the combination of the type of the cassette chromosome recombinase genes (ccr) gene complex and the class of the methicillin resistance (mec) gene complex. Besides, a rapid slide latex agglutination test (LAT) and antibiotic resistance spectrum determination before and after irradiation were performed.Results: In the strict sense, with the effect of irradiation; the presence of SCCmec subtypes IVa (22.5% vs. 10.0%), b (47.5% vs. 25.0%), & d (7.5 vs. 2.5%) or type V (15.0% vs. 7.5%) genetic elements and PVL genes (p < .001) were not proved as a signature for CA-MRSA. While, the larger SCCmec types II, and III elements were not detected in 14, and 19 from the 38, and 36 typed HA-MRSA isolates (p < .001), respectively. Remarkable effects on class A & class B mec gene complex and type2, type 3 & type 5 ccr gene complex and an increase in agglutination reaction strength in response to gamma irradiation external stimulus were observed.Conclusions: Different heterogeneous genetic composition with upregulation mecA gene expression was detected after irradiation in the HA- MRSA studied population. CA-MRSA showed remarkable ability to acquire multi-antibiotic resistance after irradiation and propose a novel paradigm for future chemotherapy against the multi-resistant pathogens whose proliferation especially among immunocompromised cancer patients is on the increase.

Staphylococcal Cassette Chromosome mec (SCCmec) Analysis of MRSA.

Methicillin-susceptible S. aureus changes to methicillin-resistant S. aureus (MRSA) upon the acquisition of staphylococcal cassette chromosome mec (SCCmec), a genomic island that encodes methicillin resistance. SCCmec elements in S. aureus are classified into different types based on the combination of mec gene complexes and ccr gene complexes, which share variations, five classes in mec and eight in ccr. To date, at least 13 types of SCCmec elements have been identified and each SCCmec type has individual characteristics. It is known that hospital-associated MRSA strains carry SCCmec elements of types, I, II, and III, and the majority of community-acquired MRSA strains carry characteristic SCCmec elements, type IV SCCmec or type V SCCmec. We herein describe multiplex PCR methods to type SCCmec elements by identifying the mec gene complex class and ccr gene complex type.

Methicillin-Resistant Staphylococcus aureus Blood Isolates Harboring a Novel Pseudo-staphylococcal Cassette Chromosome mec Element.

The aim of this work was to assess a novel pseudo-staphylococcal cassette chromosome mec (ΨSCCmec) element in methicillin-resistant Staphylococcus aureus (MRSA) blood isolates. Community-associated MRSA E16SA093 and healthcare-associated MRSA F17SA003 isolates were recovered from the blood specimens of patients with S. aureus bacteremia in 2016 and in 2017, respectively. Antimicrobial susceptibility was determined via the disk diffusion method, and SCCmec typing was conducted by multiplex polymerase chain reaction. Whole genome sequencing was carried out by single molecule real-time long-read sequencing. Both isolates belonged to sequence type 72 and agr-type I, and they were negative for Panton-Valentine leukocidin and toxic shock syndrome toxin. The spa-types of E16SA093 and F17SA003 were t324 and t2460, respectively. They had a SCCmec IV-like element devoid of the cassette chromosome recombinase (ccr) gene complex, designated as ΨSCCmecE16SA093. The element was manufactured from SCCmec type IV and the deletion of the ccr gene complex and a 7.0- and 31.9-kb portion of each chromosome. The deficiency of the ccr gene complex in the SCCmec unit is likely resulting in mobility loss, which would be an adaptive evolutionary mechanism. The dissemination of this clone should be monitored closely.

Real-time PCR followed by high-resolution melting analysis - a new robust approach to evaluate SCCmec typing of methicillin-resistant Staphylococcus aureus.

We designed a novel approach based on real-time PCR followed by high-resolution melting (HRM) analysis to determine the Staphylococcal cassette chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureusstrains, which we compared against the results of conventional multiplex PCR SCCmec typing. Multiplex PCR (for ccr and mec gene complexes) was carried out as conventional method. The HRM analysis was then designed using standard strains of each SCCmec type. The M-PCR results included types III (33.33%), IV (43.33%) and V (23.33%). HRM analysis was able to distinguish all five types, which were used to set up the protocol with a sensitivity and specificity of 100% compared with the conventional method. This novel method for SCCmec typing has high specificity and sensitivity and can be conducted in a shorter period of time at lower costs.

Methicillin-resistant Staphylococcus pseudintermedius among dogs in the description of novel SCCmec variants

The presence and genetic characteristics of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in Sri Lanka was investigated to add additional insight into global spread, emergence and evolution of MRSP. A total of 234 samples from dogs visiting veterinary clinics were cultured for staphylococci and the genomes of the MRSP isolates were sequenced, to identify resistance genes, the multilocus sequence types (MLST) and spa types. From a questionnaire the history of antimicrobial treatment and patient information was obtained.S. pseudintermedius was isolated from 116/229 samples, eight of these were MRSP. Six MRSP CC45 isolates contained a pseudo-SCC element ΨSCCmec57395. Two isolates belonging to ST429 (CC761) and ST121 (CC121) contained novel variants of the SCCmec Type V(T) element. The elements were designated SCCmecV(T)SL/066, that carried additional transposon-related genes, and SCCmecV(T)SL/154 that carried a type III restriction-modification system, a type 7 ccr gene complex, and a cadA coding sequence. Thirty-seven percent of the dogs received antimicrobial treatment at the time of sampling of which four dogs were MRSP-positive. The proportion of MRSP among S. pseudintermedius is low compared to other countries, despite the fact that in Sri Lanka antimicrobials for treatment of dogs are available over the counter. Important is the finding of novel type V(T) SCCmec elements, which further underlines the high recombination frequency of SCC elements. The ΨSCCmec57395 was found in isolates of CC45, which is the only sequence type of MRSP known to contain this pseudo-cassette.

The SfaNI restriction-modification system from Enterococcus faecalis NEB215 is located on a putative mobile genetic element.

A type IIS restriction-modification (R-M) system SfaNI from Enterococcus faecalis NEB215 has been characterized. The sfaNIM gene was cloned by the methylase selection method. Methyltransferase SfaNI, a protein of 695 amino acids, consists of two domains responsible for different DNA-strand recognition and modification, and a putative DNA-binding HTH domain located in the N-terminal part of the protein. The sfaNIR gene, located adjacent to the gene of the cognate modification methyltransferases, encodes a protein of 648 amino acids. The enzyme has been purified to apparent homogeneity and its biochemical characteristics have been described. The R-M system SfaNI is flanked by a transposase gene at its 5′ end, and a cassette chromosome recombinase ( ccr) gene complex, encoding serine recombinases CcrA and CcrB, at the 3′ end. Both proteins are specifically involved in genome rearrangement and are widely distributed among staphylococcal species. These results suggested that the R-M system SfaNI is present on the putative mobile element.

Application of the SCCmec element in the molecular typing of methicillin-resistant Staphylococcus aureus.

Acquisition of the staphylococcal chromosome cassette mec (SCCmec) is one of the key reasons for the resistance of methicillin-resistant Staphylococcus aureus (MRSA). SCCmec is composed of a mec gene complex encoding the PBP2a determinant that is responsible for the β-lactam resistance of MRSA, and a ccr gene complex encoding recombinases that mediate the integration of SCCmec into and its excision from the recipient chromosome, and so-called three junkyard (J) regions of different sizes. The SCCmec elements carried by MRSA from different geographic locations are diverse, and each type contains characteristic DNA fragments in size. These characteristics of SCCmec element may facilitate the usage of SCCmec in the molecular typing of MRSA strains. In this review, we summarize the structure and function of SCCmec elecments, and discuss the application of SCCmec elements in the molecular typing of MRSA.

The SfaNI restriction-modification system from Enterococcus faecalis NEB215 is located on a putative mobile genetic element

A type IIS restriction-modification (R-M) system SfaNI from Enterococcus faecalis NEB215 has been characterized. The sfaNIM gene was cloned by the methylase selection method. Methyltransferase SfaNI, a protein of 695 amino acids, consists of two domains responsible for different DNA-strand recognition and modification, and a putative DNA-binding HTH domain located in the N-terminal part of the protein. The sfaNIR gene, located adjacent to the gene of the cognate modification methyltransferases, encodes a protein of 648 amino acids. The enzyme has been purified to apparent homogeneity and its biochemical characteristics have been described. The R-M system SfaNI is flanked by a transposase gene at its 5(') end, and a cassette chromosome recombinase (ccr) gene complex, encoding serine recombinases CcrA and CcrB, at the 3(') end. Both proteins are specifically involved in genome rearrangement and are widely distributed among staphylococcal species. These results suggested that the R-M system SfaNI is present on the putative mobile element.